Muhammad Riaz1,2, Muhammad Hamid Rashid2, Lindsay Sawyer3, Saeed Akhtar1, Habibullah Nadeem2 and Muhammad Rizwan Javed2,

1 Department of Food Science and Technology, University College of Agriculture, Bahauddin Zakariya University, Multan, Pakistan. 2 Enzyme Engineering Lab, National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Jhang Road, Faisalabad, Pakistan. 3 Structural Biochemistry Group, Institute of Structural and Molecular Biology, The University of Edinburgh, Swan Building, Kings Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK.

Improvement in the Thermophilicity and Thermostability of Glucoamylase Through -Rays Treatment of Aspergillus niger

Abstract
Glucoamylase (exo-1,4--D-glucan glucanohydrolase: EC 3.2.1.3) yields -D-glucose from the non-reducing chain ends of a wide range of polysaccharide substrates. The operational stability of enzymes is of paramount importance for any bioprocess, which can be improved through various protein engineering techniques. Among these mutagenesis or combinatorial biosynthesis offers an easy approach to generate new enzymatic activities resulting in modified products. Local strain of Aspergillus niger was exposed to different levels of -rays (0.4-1.2 kGy) in gamma cell radiation chamber (Mark-V). Mutants (25) from 1.2 kGy exposure showed 3 log kill and were grown on 1% 2-deoxy-D-glucose containing culture plates. The best mutant was selected on the basis of glucoamylase production in submerged conditions. Glucoamylases of parent and DG-resistant mutant of Aspergillus niger were purified to apparent homogeneity. The subunit molecular mass estimated by SDS-PAGE was 93 kDa for both strains while the molecular masses determined by MALDI-TOF for wild and mutant GAs were 72.876 and 72.063 kDa, respectively. The monomeric nature of the enzymes was confirmed through activity staining. Both glucoamylases worked optimally at same pH (4.4) and temp (60 C). The pKa1 of acidic limb (proton donating group) remained unchanged (3.4) while pKa2 of basic limb (proton receiving group) of active site residues of mutant was increased (6.5) as compared to parent (6.2). The Vmax and Kcat of mutant glucoamylase were increased more than two times as compared to that of parent. The mechalis constant (Km) was decreased to 0.161 mg starch ml-1 for Aspergillus niger mutant from 0.25 mg starch ml-1 for that of parent. The specificity constant Kcat/Km for Aspergillus niger parent and mutant was 1753.39 and 5820.71, respectively. Mutant glucoamylase was better for its thermodynamic properties of substrate hydrolysis. Both of the enzymes were also studied for their behaviour in the presence of proteases and urea